The preclinical discovery and development of the combination of ivacaftor + tezacaftor used to treat cystic fibrosis

KEYWORDS: Cystic fibrosis; CFTR modulators; ivacaftor; lumacaftor; tezacaftor; elexacaftor; cellular models; clinical endpoints

1. Introduction

Cystic fibrosis (CF) is the most lethal recessive disorder in people of European descent and is caused by mutations in the gene encoding the CF transmembrane conductance reg- ulator (CFTR) protein, a chloride and a bicarbonate channel that regulates the activity of other ion channels and governs the hydration and the viscoelastic properties of mucus in several epithelia-lined tissues [1]. CFTR is a unique ATP- binding cassette anion channel [2], composed of an N-terminal lasso motif, two nucleotide-binding domains (NBDs), two membrane-spanning domains (MSDs), and a regulatory domain. In addition, the interface between the NBDs and MSDs consists of alpha-helical intracellular loops (ICLs) and coupling helices.

CF is a syndrome characterized by high sweat chloride concentration, pancreatic insufficiency, poor nutritional status, gastrointestinal symptoms, sinopulmonary disease, the last punctuated by pulmonary exacerbations, and progressive loss of lung function until respiratory failure occurs. Although all these clinical manifestations have been traced back to CFTR dysfunction as a ion channel or a regulator of other channels [3], CFTR function is involved in other cellular processes, parti- cularly a role for CFTR has been shown in the tight epithelial barrier [4] and in innate immunity responses [5]. The manage- ment of CF patients during old and novel therapies has been gauged on parameters that either evaluate directly CFTR func- tion (such as the sweat chloride concentration) or indirectly through surrogate endpoints such as FEV1 (forced expiratory volume in 1 second), a spirometric indicator of lung function, or even the nutritional status by anthropometric measures such as body mass index or body weight. Due to the preemi- nence of lung disease in both morbidity and mortality of CF people, the main effort has been directed toward endpoints evaluating the deterioration of respiratory function [6]. Very few studies have focused on noninvasive and more predictive biomarkers that could better indicate whether the applied treatment was intervening on the pathophysiological process occurring in the lung [7]. None of these, with few exceptions, have been considered in the major clinical trials that will be described in this review.

Overall, more than 2000 mutations in the CFTR gene have been discovered, of which greater than 300 are known to cause disease as described in the CFTR2 database [8]. CFTR mutations can be classified into six classes, depending on their impact on the biogenesis of the CFTR protein, its transport on the plasma membrane and its activity [9–11]. Class I mutations involve premature stop codons, that prevent full transcription of the CFTR gene and the synthesis of a truncated mRNA that is readily degraded by non-sense-mediated decay. Class II mutations have as a result the synthesis of an aberrantly folded protein that is degraded in the proteasome. The most frequent mutation in this class and in absolute is F508del, which is present on one allele in up to 90% of all CF indivi- duals, while is homozygous in almost 50% of these people [12–14]. Class III mutations lead to a channel properly inserted in the plasma membrane but that shows a gating defect. Gly551Asp (G551D) is the commonest mutation belonging to this class and which is present in approximately 4% of the patients. Class IV mutations lead to an impaired CFTR channel conductance. Class V mutations produce incorrect splice sites thus reducing the synthesis of a normal mRNA. Class VI muta- tions produce a truncated COOH-terminus of the protein lead- ing to an accelerated turnover of the protein at the apical side of epithelial cells.

Class I, II, and III are associated with a more severe phenotype (i.e. multisystemic disease with pancreatic insufficiency), whereas class IV, V, and VI present some residual function and are asso- ciated with less severe phenotype (i.e. pancreatic sufficiency) [15]. It is worth noting that this strict genotype-phenotype cor- relation is not followed when the pulmonary manifestations are considered. In this case, genetic and environmental modifiers can bring to different outcomes in the presence of the same genotype [2]. Mutations belonging to class I and II are also known as minimal function (MF) mutations since they demon- strate no to very little CFTR function (for example, G542X for class I and N1303K for class II). Mutations with residual function are those belonging to the classes IV and V and include P67L, R117C, L206W, R352Q, A455E, D579G, 711 + 3A→G, S945L, S977F, R1070W, D1152H, 2789 + 5 G→A, 327-226A→G, and 3849 + 10kbC→T. These
mutations express CFTR proteins that are processed by the endoplasmic reticulum (ER), are trafficked to the luminal surface, and either conduct some ions (class IV) or are less abundant (class V) [15–17]. Some of these mutations were associated with the so-called CFTR-related disorder (CFTR-RD), encompassing a clinical spectrum from congenital bilateral absence of vas deferens (CBAVD) to mild forms of CF [15,18]. The term CFTR- RD includes those patients with some indication of CFTR dys- function (e.g. only intermediate sweat chloride value) and only 1 CFTR mutation identified. Clinically these patients are character- ized by disseminated bronchiectasis, CBAVD, and acute or recur- rent pancreatitis. Patients compound heterozygotes for these mutations and F508del, have been more thoroughly studied and often receive a diagnosis after early infancy or later, even at adult age, present lower sweat chloride, better nutritional measures, and have a significantly lower prevalence of pancrea- tic insufficiency and CF-related diabetes than F508del homozy- gotes [19–21]. They show also better lung function and reduced infection rate with P. aeruginosa. Moreover, they have signifi- cantly lower mortality rates than patients with the classic form of CF [21].

CFTR mutations’ effetcts are not mutually exclusive, mean- ing that the same mutation can lead to multiple defects. The F508del mutation disrupts the ICL:NBD1 interface [22,23], thereby causing a processing and trafficking defect but also altering the gating of the few channels that reach the surface as well as increasing their turnover once at the cells’ surface. Overall, these combined effects result in minimal CFTR- mediated chloride transport [24–28].

2. Literature search strategy

Medline, Embase, and The Cochrane Central Register of Controlled Trials were searched for relevant studies. The key- words used were ‘CF or cystic fibrosis’ AND ‘IVA or ivacaftor’ OR ‘LUM or lumacaftor’ OR ‘TEZ or tezacaftor’ OR ‘VX-659’ OR ‘VX-445’ OR ‘elexacaftor’ OR ‘triple therapy’ OR ‘in vitro models’ OR ‘organoids’ OR ‘biomarkers’ OR ‘neutrophils’ OR ‘mono- nuclear cells’. From this search, appropriate reports on CFTR modulator therapies were identified and applicable cross- references reviewed. Recent works, appeared in the form of conference abstracts, were also reviewed by the authors per- sonally participating in these conferences.

3. CFTR modulator therapies

Traditionally, CF therapies have been focused on the manage- ment of signs and symptoms deriving from the loss of CFTR function, namely mucolytics (e.g. N-acetylcysteine) and recombi- nant human DNase for mucus obstruction [29], hypertonic saline for mucus hydration [30], antibiotics for pulmonary infections [31], as well as supplementary enzymes and nutrient and caloric intake for pancreatic insufficiency [32,33]. These therapeutic measures are effective but cumbersome [1] and have prolonged the life span of CF subjects, the median predicted age being 44 years for children with CF born in 2013–2017 [34].

A breakthrough in CF therapies has been the approval for clinical use of the so-called CFTR modulators, a class of small molecules that can allow the proper transport of class II mutated CFTR on the plasma membrane (correctors) or increase the open probability of mutated class III CFTR chan- nels already positioned on the plasma membrane (potentia- tors). Ivacaftor (formerly VX-770) is the first licensed CFTR potentiator for clinical use. Its approval by FDA in 2012 is based both on preclinical and clinical studies and now it is approved for CF people with G551D and other gating muta- tions [35]. Also, class IV mutations, determining loss of ion conductance, such as Arg117His [36], have obtained benefit from the clinical application of ivacaftor [37].

Based on the understanding of the complex molecular defects caused by the F508del-CFTR mutation, CFTR modula- tors have been utilized to increase the quantity and/or enhance the function of CFTR protein at the cell surface. Two complementary approaches have been developed to restore chloride transport for the F508del mutation. CFTR cor- rectors, such as lumacaftor (formerly VX-809) and tezacaftor (formerly VX-661) bind the F508del-CFTR protein repairing the aberrant ICL:NBD1 interfaces, thus augmenting intracellular processing and trafficking, therefore increasing the amount of mature CFTR available at the cell surface [38–42]. Moreover, it has been demonstrated that lumacaftor at high concentration (1000 μM) rescued the V232D-induced mis- folded third and fourth transmembrane helix (TM3/4), impli- cating that lumacaftor may act by enhancing the proper insertion of CFTR transmembrane segments [43]. On the other hand, the CFTR potentiator ivacaftor increases the chan- nel-gating activity of F508del-CFTR protein that is delivered to the cell surface, to augment anion transport [44,45].
Other modulators of CFTR expression and functions include [46–49]: i) stabilizers that anchor CFTR channels at the plasma membrane, thereby correcting the instability of class VI mutants (e.g. cavosonstat), ii) amplifiers that increase the amount of CFTR protein in the cell making more CFTR protein available for other therapies to work on (e.g. PTI-428), iii) molecules that bypass a specific premature termination codon (PTC) and restore mRNA levels (read-through therapies and inhibitors of nonsense-mediated mRNA decay for class I mutations), iv) antisense oligonucleotides targeted to non- canonical splicing mutations, such as the 3849 + 10 kb C-to-T splicing mutation (leading to the inclusion of a cryptic exon in the mature mRNA) in order to prevent its recognition [50], v) novel combination-potentiators (co-potentiators) displaying drug-like properties with nanomolar potency that, in synergy with Class I potentiators such as VX-770, activate CFTR harbor- ing missense, deletion, and nonsense mutations in the NBD2 [51]. Splicing mutants (e.g. −1 or +1 mutations) and large
deletions (included in class I) are still unrescuable and repre- sent altogether around 14% of all mutations [11]; therefore, there is unmet need to increase eligibility from 90% to 100% of the patients, considering also those harboring rare and ultra-rare mutations [52].

Based on these assumptions, ex vivo studies based on patient-derived material are a more feasible approach than testing every CFTR modulator for clinical ben- efit in an N-of-1 trial [53]. For example, in vitro analysis in heterologous cells confirmed in nasal epithelial cells of a patient carrying the 2909 G > A mutation, causing the glycine 970 to aspartate substitution (G970D), a very rare mutation in the Caucasians, led to the prediction that this mutation would have been highly sensitive to ivacaftor and lumacaftor, for the gating and trafficking defects, respectively, [54].

The aim of this review is to identify the major preclinical steps that have led to the evaluation and validation of ivacaf- tor and its combination with tezacaftor in comparison with the other corrector lumacaftor. Then, we revise those clinical trials which have studied the safety and efficacy of this combination therapy as well as have paved the way for triple therapies involving the combination ivacaftor–tezacaftor. Finally, we focus on the biomarker discovery and on marketing/post- marketing issues as important facets of the ivacaftor–tezacaf- tor case history.

3.1. First steps in preclinical evaluation

Ivacaftor was identified first by a high-throughput screening strategy starting from a library of 228,000 agents and lead optimization, and then validated using patch-clamp measure- ments in Fischer Rat Thyroid (FRT) cells expressing G551D- or F508del-CFTR and in primary cultures of human bronchial epithelial (HBE) cells [44,55]. In G551D/F508del HBE cells, iva- caftor was shown to potentiate CFTR-mediated chloride secre- tion approximately up to 50% of wild-type CFTR activity, whereas this effect was lower in F508del/F508del HBE cells (14% of non-CF HBE). These results were paralleled by a similar decrease in airway surface fluid (ASL) absorption and an increase in ciliary beat frequency. Overall, these results showed that new CFTR modulators could be identified in primary cultures of HBE cells, that better reflects the in vivo environment of CF patients.

The literature is not unanimous on recognizing the benefits of CFTR correctors in combination with ivacaftor. Several in vitro studies reported in the first place a negative impact of potentia- tors such as VX-770 on the correction of F508del-CFTR matura- tion defect by lumacaftor or tezacaftor, suggesting an interference between potentiators and correctors. In 2014, a study by Cholon et al. [56] was conducted on primary CF HBE cultures (F508del/F508del), which were treated with a corrector (tezacaftor or lumacaftor) with acute or chronic treatment of ivacaftor (1–5 μM). Subsequently, Ussing chamber experiments were performed to measure F508del-CFTR function in the pre- sence of amiloride, forskolin, and CFTR inhibitor-172 (CFTRInh172). The results of this experiment highlighted how chronic ivacaftor treatment caused a dose-dependent reversal of lumacaftor or tezacaftor-mediated CFTR correction in primary F508del homo- zygous HBE cultures. To better explore the mechanism mediat- ing the ivacaftor-induced reduction of lumacaftor-corrected F508del-CFTR function, Western blotting techniques were used to analyze protein maturation and turnover. Chronic treatment with lumacaftor alone resulted in a rescue of mature band C which was not present in vehicle- or ivacaftor-treated primary F508del-CFTR HBE cells. However, when CF cells were treated chronically with both drugs, the amount of mature F508del-CFTR was diminished, and the F508del-CFTR protein appeared almost entirely as immature band B, suggesting that under these experimental conditions the reduced capacity for Cl− secretion resulted
in an increased turnover rate of corrected F508del-CFTR. Overall, these findings revealed that chronic treatment with CFTR poten- tiators and correctors may have unanticipated effects that can- not be predicted from short-term studies. Veit et al. published a similar study in which they treated immortalized bronchial epithelial cells (CFBE41o- line) with ivacaftor at 1–1000 nM and lumacaftor or tezacaftor at 3 µM [57]. Different assays were used to evaluate the effect of prolonged exposure to ivacaftor in presence of either corrector. Ivacaftor treatment for 24 h decreased the amount of the F508del mature band (C-band) in CFBE lysates in a dose-dependent manner. The ivacaftor effect was attenuated in lumacaftor- or tezacaftor-treated cells, prob- ably due to the partial stabilization of the mature F508del-CFTR pool by the corrector. Additionally, they performed short-circuit current measurements after 24 h of incubation with 100 nM ivacaftor and 3 µM lumacaftor or tezacaftor in both CF and prim- ary HBE cells. The outcome of these experiments showed a decrease of F508del-CFTR chloride conductance after a long- term exposure to ivacaftor. The authors affirmed that long exposure to ivacaftor diminished the folding efficiency and the metabolic stability of F508del-CFTR at the ER and post-ER com- partments, causing, respectively, reduced cell surface F508del- CFTR density and function.

On the other hand, Matthes et al. [58] showed that chronic exposure to ivacaftor with clinically relevant concentration (1–100 nM) does not diminish the functional expression of rescued F508del-CFTR by lumacaftor in primary F508del-HBE cells. The authors suggested that the therapeutic benefit of ivacaftor plus lumacaftor is limited by the efficacy of lumacaf- tor rather than by ivacaftor. Furthermore, they also demon- strated, in line with the work of Veit et al. [57], that an extended exposure to 100 nM ivacaftor plus 1 µM lumacaftor during the pre-treatment period (24 h) reduced by ~40% the subsequent functional response to acute 10 µM ivacaftor plus 10 µM forskolin in respect to the strong response obtained when cells had been pre-treated with lumacaftor alone. Interestingly, no decrease in functional expression was observed after prolonged exposure to 1–100 nM levels of ivacaftor when function was assayed as the response to for- skolin alone or to forskolin plus ivacaftor (1–100 nM).

More recently, Chin et al. [59] observed that chronic (48 h) micromolar concentration of ivacaftor (10 µM) has a negative effect on the stabilization of the mature band C form of the 3 µM lumacaftor-rescued F508del-CFTR protein and it also reverses lumacaftor-mediated enhancement of the interaction between the intracellular loop conferred by MSD2 (ICL4) and NBD1. Interestingly, ivacaftor exerted a similar negative effect on the stability of other membrane-localized solute carriers (SLC26A3, SLC26A9, and SLC6A14), suggesting that this negative effect is not specific for F508del-CFTR. To be noticed, the treatment of F508del- CFTR with a panel of ivacaftor derivatives showed that the degree of ivacaftor lipophilicity is correlated to its destabilizing effect. The nonspecific effect of chronic ivacaftor on the steady-state abun- dance of F508del-CFTR, other membrane proteins, and lipid rafts was observed at the suprapharmacological concentration of 10 µM. According to Matthes et al. [58], a peak of free plasma concentration of ivacaftor is 1.5–8.5 nM, considerably lower than other estimates (Vertex, New Drug Applications 203188 and 206038, U.S. Food and Drug Administration, 2012 and 2015), and lower than the concentration at which Chin et al. [59] observed the nonspecific effects of ivacaftor. However, as noted previously [60], there was a wide range of serum ivacaftor concentration in different patients, though almost always below 10 µM.

A recent study by Borcherding et al. [61] was conducted in F508del homozygous CF HBE cells with a tezacaftor (1 and 10 µM) and ivacaftor (1 µM) combined treatment for 48 h. The research showed a very little stimulated F508del- CFTR current due to the presence of ivacaftor.
Differently, other studies have underlined the beneficial consequence of ivacaftor and lumacaftor or tezacaftor drug combination in vitro. Van Goor et al. [45] discovered that acute application of ivacaftor increased the lumacaftor-corrected F508del-CFTR-mediated short-circuit current (Isc) across a monolayer of cultured HBEs (to a level of ∼25% of wild-type HBE) and further increased ASL height in these cells. The obtained data supported the rationale of combining CFTR correctors and potentiators as one strategy for CF therapy.

In 2013, at the 36th European CF conference, Donaldson and colleagues stated that tezacaftor increases F508del-CFTR protein activity in vitro both alone and in combination with ivacaftor and enhance F508del-CFTR trafficking to the cell surface [62]. In 2016 Van Goor and colleagues presented two different experimental approaches [63]. The first one was car- ried out by Western blot studies and the second one by Ussing chambers studies. Both were conducted on primary HBE cells, derived from homozygous F508del-CF donors, treated for 24 h with 3 µM tezacaftor either alone or in combination with 100 nM ivacaftor. The in vitro data obtained demonstrated that the combination of tezacaftor and ivacaftor improved the F508del-CFTR expression in membrane and consequently enhanced chloride transport more than either drug alone.
In 2018, the Brochiero research group showed that the combination lumacaftor (5 μM) plus ivacaftor (1 μM) efficiently rescued cAMP-activated and CFTRInh172-sensitive Cl− currents on polarized and differentiated primary airway epithelial cell cultures from patients carrying the F508del mutation, but also highlighted a positive effect by the combination of the two molecules on airway epithelial repair [64] and in reducing airway inflammation [65].

As aforementioned, the combinations lumacaftor–-ivacaftor and tezacaftor–ivacaftor show modest CFTR correction com- pared to wild-type CFTR; therefore, there is an unmet need to identify more efficacious correctors of CFTR that will translate into improved clinical benefit for CF patients. It has been demonstrated that CF therapies which correct CFTR domain assembly, in particular by stabilizing NBD1-MSD1/2, represent a useful strategy for correcting F508del-CFTR trafficking to the plasma membrane [66–68]. These second-generation correc- tors, VX-445 (now elexacaftor) and VX-659, designed to rescue function of F508del-CFTR, have different mechanisms of action compared to first-generation correctors. In 2018, two papers have analyzed the benefits of a three drug combination (two correctors and one potentiator) in vitro. In the first report, Keating et al. [69] have shown that the CFTR corrector VX- 445 (2 μM) increased expression of mature CFTR protein in HBE cells isolated from four donors with F508del–MF geno- types and three donors with the F508del/F508del genotype. The combination of VX-445 and tezacaftor (18 μM), with or without ivacaftor (1 μM), increased levels of mature CFTR protein and led to an increase in chloride transport that was greater than that in cells exposed to VX-445 or tezacaftor alone.

The second report by Davies et al. [70] confirmed that the combination of tezacaftor (18 µM) with ivacaftor (1 µM) defined an increase both of the mature band of F508del- CFTR (C-band) in membrane and of the F508del-CFTR activity in HBE cells derived from patients with F508del-MF or F508del- F508del genotypes. While treatment with VX-659 alone pro- duced a modest increase in Cl− transport, the combination of VX-659–tezacaftor–ivacaftor produced the greatest increase in Cl− transport.

3.2. CF patient-specific tissue model for preclinical drug testing

The abovementioned discrepancies among studies may reflect the inappropriateness of in vitro models when the F508del mutation has to be corrected in patients. The combination therapies – lumacaftor–ivacaftor (prescribed as Orkambi®) and tezacaftor–ivacaftor (prescribed as Symdeko®) – have been approved by the FDA for patients bearing the F508del CFTR mutation. Despite data showing that these therapies can rescue F508del-CFTR function to approximately 15% of normal channel activity in human bronchial epithelial cells [45], Orkambi® and Symdeko® therapies are associated with modest clinical responsiveness for patients homozygous for the F508del mutation [71,72] (see Section ‘Clinical development’ for details). Discrepancies between in vitro studies and clinical outcomes emphasize the need for the development of better models to be used for testing clinical compounds. To date, baby hamster kidney (BHK-21), FRT, human embryonic kidney (HEK293) and CFBE41o- immortalized cell lines have been used to screen CFTR modulators; however, these models have demonstrated that heterologous expression systems do not always predict CFTR modulator efficacy in vivo [73–76]. Recently, the North American Cystic Fibrosis Foundation developed 16HBE14o- immortalized bronchial epithelial cells edited using CRISPR-Cas9 to introduce CFTR mutations (i.e. F508del, G542X and W1282X) in the context of the complete CFTR gene [77]. This edited cell line can be polarized and could be used to test the effect of novel CFTR modulators on CFTR channel function. Although primary bronchial epithe- lial cells are considered a good model for evaluating the efficacy of modulator therapies, they are currently limited by the availability of biopsy specimens, damage occurring in the primary site, and poor expansion potential [78]. More recently, rectal biopsies and nasal epithelial brushings have been devel- oped as a CF patient-specific tissue models for preclinical drug testing [68,79–85].

Primary human nasal epithelial (HNE) cells, obtained with a simple brushing of nasal cavities, may be more affordable in terms of availability. HNE cells confer the relative advantage of modeling the epithelium in the airways. Interestingly, Brewington et al. demonstrated that brushed HNE cell cultures recapitulate the functional CFTR characteristics of HBE with fidelity and are therefore an appropriate noninvasive HBE surrogate as a model in preclinical studies [86]. Recently, Pranke and colleagues showed that CFTR chloride channel responses observed in patient-derived nasal epithelial cultures correlated with individual’s improvement in FEV1, measured after initiation of Orkambi® treatment [82]. However, as stated by the authors, there was a great inter-patient variability, indicating that further development in this technique is needed.

Overall, these limitations may be addressed by the use of patient-derived cells that can organize in tissue-like struc- tures [87]. Patient-derived intestinal organoids recapitulate human epithelial biology and patient-specific characteristics, like the expression of intronic and intergenic enhancers which regulate CFTR gene expression and have been used for disease modeling [83]. Advantages of this gastrointest- inal-based system are that the cells are obtained from stable individuals through an endoscopy or suction biopsy proce- dure. Moreover, organoids are sensitive to modulator effects and have a large dynamic functional readout [46]. The in vitro CFTR functional assay in patient-derived intestinal organoids termed forskolin-induced swelling (FIS assay) is based on adenylyl cyclase activator-mediated CFTR channel activation via increased cyclic AMP (cAMP) concentrations. CFTR is expressed on the apical membrane and therefore localized to inner membrane in this organoid system; there- fore, cAMP stimulation by forskolin leads to ion and fluid transport into the organoid lumen, causing swelling. Through the FIS assay, CFTR activity can be determined by quantifying the amount of fluid secreted into the lumen by measuring changes in the size of the organoid lumen after adding the adenylyl cyclase activator forskolin [83,85,88,89]. Interestingly, three independent laboratories among Leuven, Lisbon, and Utrecht showed high between-lab simi- larity (>95%) in organoids swelling from the same six CF patients with distinct CFTR genotypes, treated with ivacaftor and/or tezacaftor (VX-661) [90]. Importantly, Beekman and colleagues found a correlation similar to what it was found in HNE between the rescue of CFTR-mediated rectal orga- noid swelling for F508del and rare mutations with in vivo response to indicators of CFTR modulators (change in FEV1 and sweat chloride concentration), allowing for predictions to be made about CF patient responses to drug therapies from preclinical data [85,91].

Intestinal organoids, as well as airway and pancreatic organoids, can be derived from induced pluripotent stem cells (iPSC). It has been demonstrated that CF iPSC-derived organoids showed defects in forskolin-induced CFTR depen- dent swelling and can be rescued by CFTR modulators or gene editing to correct the CFTR mutation to wild-type CFTR [92,93].

Airway spheroids, both bronchial and nasal, can be also derived from primary airway basal cells [94]. Interestingly, while Brewington et al. [95] demonstrated that CFTR is expressed at the apical side of the inner membrane in the nasal cell spheroids, and forskolin stimulation induced swel- ling, Guimbellot et al. [96] showed that CFTR is expressed on the apical side of the nasal spheroids face outward. Therefore, in non-CF nasospheroids, CFTR stimulation by forskolin, induced ion and fluid movement from the lumen to the exterior bath causing reduction in cross-sectional area. However, no changes in size were observed in CF spheroids. Treatment of nasospheroids homozygous for F508del with Orkambi®, showed a significant shrink in size, indicating phar- macologic rescue of F508del-CFTR function [96].
Overall, airway spheroids could be used as a predictive tool to enable preclinical trials to identify CFTR modulators for personalized medicine for CF patients.

4. Clinical evaluation

Based on the positive preclinical test results, ivacaftor was swiftly advanced into clinical trials (later available for patients as Kalydeco®). The results of phase 2 and 3 clinical trials concerning ivacaftor have been assessed in previous reviews and we refer the reader to this literature [97,98]. Here, we will highlight some results that would be interesting in the light of evaluating tezacaftor as monotherapy and as double and triple therapies in CF patients.

4.1. Ivacaftor in monotherapy and double therapy with lumacaftor

Ivacaftor safety was first evaluated in a phase 2 clinical trial in adult CF subjects with at least one copy of G551D mutation [99] providing support for further clinical evaluation concerning its efficacy. Subsequently, two 48-week randomized, double-blind, placebo-controlled phase 3 clinical trials examined ivacaftor monotherapy in patients with CF possessing the G551D CFTR mutation, demonstrating rapid and sustained improvement in clinical outcome measures and CFTR biomarkers with an accep- table safety profile [100,101]. The study by Ramsey and collea- gues [100], called STRIVE, evaluated ivacaftor (150 mg every 12 h) in CF patients with G551D CFTR 12 years of age and older with a percent predicted FEV1 (ppFEV1) of 40–90%. The study by Davies et al. [101], called ENVISION, was conducted in children aged 6–11 years with ppFEV1 measurements of 40–105%. The primary efficacy endpoint in both trials was the absolute change from baseline to week 24 in ppFEV1. Patients on ivacaftor in the STRIVE study showed a sustained increase in ppFEV1 of 10.6 percentage points (10.4% points with ivacaftor, −0.2% points with placebo; p < 0.001). In the ENVISION study, the relative increase from baseline was 12.5 percentage points in the ppFEV1 in the ivacaftor group (12.6% points with ivacaftor, 0.1% points with placebo; p < 0.001). Moreover, in both studies, patients on ivacaftor gained more weight, had an increase in scores on the Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain, indicating a reduction in respiratory symptoms (although not significantly in the ENVISION study), and presented a significant reduction in sweat chloride concentrations, that often fell within the normal range. Refer to Table 1 for compar- ison among these studies. Ivacaftor was also tested in a 24-week, open-label phase 3 trial (KIWI study) in children aged 2–5 years with at least one CFTR gating mutation, that demonstrated that the pharmaco- kinetics, safety, and efficacy were generally similar to those seen in older patients [102]. KLIMB was an 84-week, open-label extension of KIWI, that assessed that the improvements in sweat chloride, weight, body mass index (BMI), fecal elastase- 1 observed during KIWI were maintained during this extension study [103]. The increase in fecal elastase-1 concentrations was also observed in a subsequent 24 weeks-phase 3 trial of ivacaftor in children 12 to <24 months, suggesting that ivacaftor could potentially preserve pancreatic function, if initiated early in life [104]. In small children on ivacaftor treatment, asymptomatic transaminase elevation occurred more frequently than in studies of ivacaftor in older patients (in up to 30% of cases), but very rarely required permanent treatment discontinuation [102–104]. Ivacaftor was subsequently used also in F508del homozy- gous patients as double therapy in conjunction with the cor- rector lumacaftor (Orkambi®). This combination was the consequence of the lack of clinical efficacy of ivacaftor mono- therapy [105], due to few activatable CFTR proteins on the plasma membrane, and also of lumacaftor monotherapy in these patients [106], due to the insufficient activity of the mutated F508del protein once properly positioned on the plasma membrane. Two phase 3 randomized control trials, termed TRAFFIC and TRANSPORT, analyzed the efficacy of adding ivacaftor to potentiate the function of the lumacaftor- rescued CFTR in patients 12 years of age or older who were homozygous for the F508del CFTR mutation [71]. The main results of this study are reported in Table 1. The difference between ivacaftor–lumacaftor and placebo with respect to the mean relative change in ppFEV1 was significant and ranged from 4.3% to 6.7%, although lower than that of ivacaftor monotherapy in G551D patients. Patients treated with luma- caftor–ivacaftor had significant improvements in BMI and CFQ- R score as compared with placebo, and also reductions in the rate of pulmonary exacerbations. Importantly, patients in the treatment arms reported more adverse events that led to the discontinuation of the study and dyspnea and chest tightness were reported more frequently in the active-treatment groups. Moreover, serious adverse events related to the elevation of liver enzymes were reported only in the active-treatment groups, although only in seven patients. Other trials have evaluated safety and tolerability of ivacaf- tor–lumacaftor in various populations of F508del homozygous patients across different ages and lung disease severity [107– 112]. In summary, some general issues can be derived: (1) adverse respiratory events were largely associated with treat- ment initiation [107] but with less frequency in children less than 12 years of age [110–112]; (2) the increase in ppFEV1 was of minor entity [110,111]; (3) patients with severe lung disease may benefit from treatment initiation at a lower dose with close monitoring before subsequent increase to the full dose [107]; (4) in younger patients with normal spirometry, lung clearance index (LCI), a measure of ventilation inhomogeneity, is more sensitive to detect early lung damage associated with CF, characterized by peripheral rather than central airway impairment assessed by spirometry [110,111] and also to monitor the response to therapy [111]. Overall, although the combination ivacaftor–lumacaftor has been a milestone for patients homozygous for the F508del mutation, encompassing around 45% of CF patients, this dou- ble therapy is a cause of concern for both efficacy and safety. First of all, a less unfavorable drug-to-drug interaction of that presented by ivacaftor–lumacaftor is needed [113,114]. Respiratory side effects and multiple drug interactions (includ- ing hormonal contraceptives and rifampin) limit the use of this combination therapy [71,115]. The introduction of novel- generation correctors, such as tezacaftor, in double or triple therapies is finalized to improve both satety and efficacy of the CFTR modulators in patients harboring class II mutations. 4.2. Tezacaftor in monotherapy and double therapy with ivacaftor One randomized, placebo-controlled, double-blind phase 2 study [72] evaluated the safety and efficacy of tezacaftor monotherapy and of ivacaftor–tezacaftor combination therapy (later available for patients as Symdeko® in the USA and Canada, and Simkevi® in Europe). Patients homozygous for F508del received tezacaftor (10 to 150 mg) every day alone or in combination with ivacaftor (150 mg every 12 h) in a dose-escalation phase, as well as in a dosage regimen test- ing phase. Subjects compound heterozygous for F508del and G551D, taking physician-prescribed ivacaftor, received tezacaf- tor (100 mg every day). Primary endpoints were safety through Day 56 and change in sweat chloride from baseline through Day 28, while secondary endpoints were changes in ppFEV1 from baseline through Day 28 and pharmacokinetics. Tezacaftor (100 mg every day) in combination with ivacaftor (150 mg every 12 h) was selected during the dose-escalation phase as the most effective dose (Table 2). In subjects homo- zygous for F508del, ivacaftor–tezacaftor combination therapy resulted in a 6.04 mmol/L decrease in sweat chloride (treat- ment efficacy of 5.18 given that placebo group showed a decrease of 0.86 from the baseline; p < 0.05) and 3.75 per- centage point increase in ppFEV1 (treatment efficacy of 3.89, since the placebo group presented a decrease of 0.14 from the baseline; p < 0.05). In patients F508del-G551D compound het- erozygous, it was observed a 7.02 mmol/L decrease in sweat chloride (treatment efficacy of 17.20, with the placebo group having an increase of 10.18 over the baseline; p < 0.05) and 4.60 percentage point increase in ppFEV1 (treatment efficacy of 3.20, due to the increase of 1.40 in the placebo group from the baseline; p < 0.05). The incidence of serious adverse events was lower in the tezacaftor monotherapy and tezacaftor–iva- caftor combination groups than in the placebo group, due largely to the numerically lower number of pulmonary exacer- bations. After discontinuation of treatment, sweat chloride and lung function values returned to near pretreatment levels (Day 28 to Day 56), providing further evidence that the effects observed during active dosing were treatment related. Moreover, improvements in lung function in patients homo- zygous for F508del were generally comparable to or numeri- cally greater than those observed in patients treated with lumacaftor–ivacaftor in phase 3, 24-week TRAFFIC/ TRANSPORT studies [71]. This study showed that ivacaftor– tezacaftor combination therapy is well tolerated, with low rates of discontinuation and similar incidences of adverse events in the study groups. Importantly, treatment initiation with ivacaftor–lumacaftor is sometimes associated with respiratory events and acute lung function decline [71,108,116,117], making the improved benefit-to-risk profile of tezacaftor–ivacaftor a potential treatment option for a greater proportion of patients homozygous for F508del. As to the pharmacokinetic parameters, exposures of ivacaftor and its metabolites, M1 and M6, were unaffected by the co- administration with increasing doses of tezacaftor and were consistent with previously observed exposures [118]. Compound F508del/G551D patients also showed an improvement in sweat chloride concentrations and ppFEV1 after ivacaftor–tezacaftor treatment in comparison with pla- cebo-treated (only ivacaftor) patients, suggesting the potential for the efficacious treatment of those patients harboring F508del and an ivacaftor-responsive mutation [72]. Subsequently, one phase 3, randomized, double-blind, pla- cebo-controlled trial evaluated combination therapy with iva- caftor and tezacaftor, called EVOLVE, involved patients 12 years of age or older who were F508del homozygous [119]. The treatment regimen (100 mg of tezacaftor once daily and 150 mg of ivacaftor twice daily) was chosen on the basis of the phase 2 trial. The primary endpoint was the absolute change in ppFEV1 through week 24. Key secondary endpoints were the relative change in ppFEV1, the number of pulmonary exacerbations, the absolute change in the BMI, and the absolute change in the respiratory domain score on the CFQ-R, as well as the incidence of adverse events. The use of tezacaftor–ivacaftor led to a significantly greater absolute change from baseline in the percentage of ppFEV1 than placebo (4.0 percentage points). The mean absolute change from baseline through week 24 was 3.4 percentage points in the tezacaftor–ivacaftor group, as compared with −0.6 percentage points in the placebo group (p < 0.001). The difference vs. placebo in the relative change in ppFEV1 through week 24 was 6.8% (−0.5 in the placebo group and 6.3 in the treatment group; p < 0.001). This effect was rapid in onset, was sustained throughout the trial, and was observed in all the prespecified subgroups. Tezacaftor–ivacaftor was asso- ciated with a significantly lower frequency of pulmonary exacerbations than placebo (Table 2), as well as a longer time to the first exacerbation and a lower rate of exacerba- tions that led to hospitalization or the use of intravenous antibiotics. No significant differences in BMI were observed (Table 2). The use of tezacaftor–ivacaftor led to an improve- ment in the respiratory domain scores on the CFQ-R (Table 2), suggesting that treatment resulted in improved quality of life in these patients. In addition, the use of tezacaftor–ivacaftor led to a rapid and sustained reduction in the sweat chloride concentration (Table 2). The incidence of adverse events was similar in the tezacaftor–ivacaftor and the placebo groups. Serious adverse events were reported in 31 patients (12.4%) in the tezacaftor–ivacaftor group and in 47 (18.2%) in the placebo group. The rate of respiratory adverse events was not higher in the tezacaftor–ivacaftor group than in the pla- cebo group, indicating that the safety profile for tezacaftor– ivacaftor is better than that reported for lumacaftor–ivacaftor. A phase 3, randomized, double-blind, placebo-controlled crossover trial, called EXPAND (Table 2), evaluated the efficacy and safety of tezacaftor–ivacaftor combination therapy and iva- caftor monotherapy in patients aged 12 years or older with CF heterozygous for F508del-CFTR and a residual function-CFTR mutation [120]. The primary endpoint was absolute change in ppFEV1 from study baseline to the average of the week 4 and week 8 measurements. The key secondary endpoint was abso- lute change in CFQ-R respiratory domain score, while additional secondary endpoints included relative change in percentage of ppFEV1 and absolute change in sweat chloride, from study baseline to the same time frame. The treatment difference versus placebo in the absolute change in ppFEV1 from study baseline to the average of week 4 and week 8 was 6.8 percen- tage points (p < 0.001) for tezacaftor–ivacaftor and 4.7 percen- tage points (p < 0.001) for ivacaftor, while the difference vs. placebo in the relative change in ppFEV1 was greater and is reported in Table 2. Moreover, the treatment difference between tezacaftor–ivacaftor and ivacaftor was statistically sig- nificant in favor of tezacaftor–ivacaftor (p < 0.001). Both tezacaf- tor–ivacaftor and ivacaftor were associated with statistically significant improvements compared with placebo in the key secondary endpoint absolute change in CFQ-R respiratory domain score (Table 2), and improvements were observed in both active treatment groups vs. placebo for other secondary endpoints. There were no discontinuations in the tezacaftor– ivacaftor group, two (1.3%) patients in the ivacaftor group, and one (0.6%) patient in the placebo group. To be noted, important adverse respiratory symptoms such as acute bronchoconstric- tion or ppFEV1 decrease within 2–4 h after tezacaftor–ivacaftor or ivacaftor administration were not observed, a finding distinct from lumacaftor-based regimens [71,121]. As in the case of lumacaftor–ivacaftor, also the combina- tion tezacaftor–ivacaftor has been tested in CF children aged 6 through 11 years [122]. A phase 3, open-label study eval- uated the pharmacokinetics, safety, tolerability, and efficacy of tezacaftor–ivacaftor in patients who were homozygous for the F508del mutation or had 1 copy of the F508del-CFTR mutation and an eligible residual function mutation. Part A of the study established the dose according to the weight, while part B assessed safety and tolerability as determined by treatment-emergent adverse events (TEAEs), and efficacy measurements. In part A, the exposures of tezacaftor, ivacaf- tor and their metabolites were within the range the target range of exposures (AUC: area under the curve) for patients aged 12 years or more. In part B, most children had TEAEs that were mild (48,6%) or moderate (40.0%) in severity, and no life-threating TEAEs or deaths occurred. Seven children (10%) had elevations in transaminases >3x the upper limit of normal (ULN) on ≥1 occasion, and 6 children (8.6%) had adverse events of elevated transaminases of mild severity. The treatment with tezacaftor–ivacaftor reduced sweat chloride concentration (−14.5 mmol/L from the baseline) that was rapid and sustained over 24 weeks. The mean absolute change from baseline in CFQ-R respiratory domain child version score through 24 weeks was 3.4 points. The absolute and relative change from baseline in ppFEV1 through week 24 were 0.9 percentage points and 1.4%, respectively. Growth parameters (weight, height, and BMI) were close to normal for age at baseline, and normal growth was maintained over 24 weeks of treatment.

Table 2 reports the main findings of phase-2 and phase-3 studies involving tezacaftor in monotherapy and double ther- apy with ivacaftor.
As it can be seen from the above cited results obtained in clinical trials, although the combination tezacaftor–ivacaftor does not have efficacy advantages over lumacafor–ivacaftor, its use should be recommended due to a better side effect profile and also less frequent drug interactions than lumacaftor–ivacaftor. Respiratory tightness and dyspnea were not noted with tezacaftor–ivacaftor initiation as had been with lumacaftor–ivacaftor [119,120]. Moreover, this combined ther- apy does not interfere with hormonal contraception, although the use of rifampin and other strong CYP3A inducers is not recommended [123].

A Cochrane intervention review on the correctors in CF individuals with at least one F508del mutation [124] concluded that combination therapies (lumacaftor–ivacaftor and tezacaf- tor–ivacaftor) each result in similarly small improvements in clinical outcomes; specifically, improvements in quality of life (moderate-quality evidence), in respiratory function (high- quality evidence) and lower pulmonary exacerbation rates (moderate-quality evidence). While lumacaftor–ivacaftor is associated with an increase in early transient shortness of breath and longer-term increases in blood pressure (high- quality evidence), tezacaftor–ivacaftor treatment was not asso- ciated with these adverse effects.

4.3. Tezacaftor in triple therapy

The outcomes of lumacaftor plus ivacaftor and tezacaftor plus ivacaftor in CF patients homozygous for the F508del mutation showed improvements in lung function (2.6–4.0 percentage points of ppFEV1) and decreases in the rate of pulmonary exacerbations (with a reduction of 35–39%) [71,119]. New correctors with a mechanism of action different from that of the first-generation corrector tezacaftor were analyzed for the purpose of augmenting the amount of F508del-CFTR protein at the cells’ surface and that would be amenable to ivacaftor activation in F508del homozygous patients. Moreover, triple therapy was envisioned to be valuable in the efficacious treat- ment of those patients who are heterozygous for the F508del mutation and a minimal-function CFTR mutation (F508del-MF CFTR genotypes), which represents the 30% of total patient population without a treatment addressing the basic defect [125,126]. The two small molecule drugs VX-659 and VX-445 were investigated first in phase 2 trials [69,70].

After a phase 1 trial in healthy volunteers and a small cohort of F508del-MF patients (n = 12), a randomized, placebo- or active-controlled, double-blind, dose-ranging, phase 2 trial was conducted with multiple dose levels of VX-659 [70]. This trial enrolled patients 18 years or older with a F508del-MF or F508del/F508del CFTR genotypes. Patients with F508del-MF genotypes received 4 weeks of VX-659 at doses of 80, 240, or 400 mg once daily together with ivacaftor (150 mg every 12 h) and tezacaftor (100 mg once daily) or triple placebo. Patients homozygous for F508del mutation received 4 weeks of ivacaftor–tezacaftor during a run-in period, followed by 4 weeks either on VX-659 (400 mg once daily) or matched placebo in combination with ivacaftor–tezacaftor. This inter- vention period was followed by a 4-week washout period of VX-659 (ivacaftor–tezacaftor only). The third part of the trial involved patients with F508del-MF genotypes who received VX-659 and tezacaftor together with VX-561 that is a deuterated form of ivacaftor. Replacement of hydrogen with deuterium prolongs the metabolism of the compound, allowing for once-daily dosing.

All doses of triple treatment resulted in significant improve- ment in ppFEV1 as compared with the baseline in patients with F508del-MF or F508del-F508del genotypes (Table 3). The relative change of ppFEV1 with the highest dose of VX-659 in patients with the F508del/MF genotypes was 24.6 percentage points. The increases of ppFEV1 were observed on Day 15 and were sustained up to Day 29. Improvements in sweat chloride concentration and the CFQ-R respiratory domain score were observed (Table 3). Similar improvements in all the endpoints were observed in patients with F508del-MF genotypes who received VX-659–tezacaftor–VX-561.

VX-445 was tested in a three-part, randomized, double- blind, placebo- or active controlled trial [69]. Patients with F508del-MF genotypes were assigned to receive 4 weeks of active treatment (VX-445 (50, 100, or 200 mg) in triple combi- nation with tezacaftor (100 mg per day) and ivacaftor (150 mg every 12 h)) or a triple placebo control. Another cohort of patients with F508del-MF genotypes received VX-445, tezacaf- tor and VX-561. Patients homozygous for the F508del mutation received a 4-week run-in treatment with ivacaftor–tezacaftor and then were assigned to receive 4 weeks of treatment with either VX-445–tezacaftor–ivacaftor combination or matched placebo plus tezacaftor and ivacaftor.

Treatment with VX-445-teazacaftor–ivacaftor resulted in sig- nificant improvements over baseline in ppFEV1 in patients with F508del-MF genotypes and those with the F508del/ F508del genotype (Table 3). As compared with placebo, in F508del-MF patients, the treatment efficacy for ppFEV1 was 19.0, 13.5, and 25.9 percentage points for 50, 100 and 200 mg VX-445, respectively, and in F508del/F508del patients 17.8 percen- tage points. In the treatment groups, the improvement in ppFEV1 was observed at the first assessment on Day 15 and maintained at Day 29. In parallel, improvements in CFQ-R scores and sweat chloride concentration were observed. For CFQ-R, in F508del-MF patients, the treatment efficacy was 17.2, 14.5, and 21.3 points for 50, 100, and 200 mg VX-445, respectively, and in F508del/F508del patients, the treatment efficacy was 16.3 percen- tage points. Treatment efficacy for sweat chloride was −36.0,−31.0, and −36.9 mmol/L in F508del-MF patients for 50, 100, and 200 mg VX-445, respectively, and −37.4 in F508del/ F508del patients. Patients on VX-445–tezacaftor–VX561 had similar improvements in efficacy outcomes.

Collectively, VX-445–tezacaftor–ivacaftor triple therapy has an acceptable adverse-event profile in both populations stu- died. The addition of VX-445 to ivacaftor–tezacaftor resulted in a higher clinical efficacy than tezacaftor–ivacaftor dual therapy.
No concerning safety issues across the genotype groups and range of VX-659 triple combination were observed. There were no discontinuations of the trial regimen in any patient who received VX-659–tezacaftor–ivacaftor. VX-445 triple com- bination treatment brought to discontinuation as a result of adverse events in 8% of the patients. Overall, the safety profile of both VX-659 and VX-445 was similar to that observed for the combination of tezacaftor and ivacaftor, a finding that supports further development of the two triple combinations. Two phase 3 randomized, double-blind trials have recently assessed efficacy and safety of VX-445 (now elexacaftor) in combination with tezacaftor and ivacaftor in CF individuals either homozygous for the F508del mutation [127] or with the F508del-MF genotype [128].

In the trial involving F508del homozygous patients [127], after an initial 4-week tezacaftor plus ivacaftor run-in period, subjects were randomly assigned (1:1) to receive either elezacaftor (200 mg every 24 h) plus tezacaftor (100 mg every 24 h) plus ivacaftor (150 mg every 12 h) or tezacaftor plus ivacaftor. The primary outcome was the absolute change from baseline (mea- sured at the end of the run-in period) in ppFEV1 at week 4. Secondary outcomes were absolute change in sweat chloride and CFQ-R score. Patients on triple therapy showed improve- ments in ppFEV1 of 10.0 percentage points and in the key secondary outcomes of sweat chloride concentration (−45.1 mmol/L), and CFQ-R respiratory domain score of 17.4 points, compared with tezacaftor–ivacaftor group. Importantly, the mean value of sweat chloride was below the diagnostic threshold for CF (48 mmol/L vs. 60 mmol/L). The absolute increase of 16.0 points in the CFQ-R score with triple therapy largely exceeds the 4-point improvement corresponding to the minimal clinically important difference in those CF individuals with stable disease. Although limited in time (4 weeks), the treatment with triple therapy resulted also in the improvement of parameters related to the nutrition status: there was an increase in BMI of 0.60 kg/cm2 and of body weight of 1.6 kg, compared with tezacaftor plus ivacaftor. The triple combination was well tolerated, with no discontinuation, and with very few (4%) serious adverse events (with 2% of participants in the tezacaftor–ivacaftor arm).

In the larger phase 3 trial involving patients with the F508del/MF genotypes, subjects received triple therapy for 24 weeks against placebo [128]. The primary endpoint was absolute change from baseline in ppFEV1 at week 4. Key secondary endpoints were absolute changes from baseline in ppFEV1 through week 24, num- ber of pulmonary exacerbations through week 24, sweat chloride at week 4 and through week 24, CFQ-R respiratory domain score through at week 4 and through week 24, BMI at week 24. Treatment with triple therapy resulted in significant improvement in absolute change from baseline of ppFEV1 both at 4 weeks and also at week 24 (Table 3). Triple therapy was associated with a 63% lower annualized rate of pulmonary exacerbations than placebo. Sweat chloride concentration and the CFQ-R score improved sig- nificantly already at 4 weeks and this effect maintained through week 24 (Table 3). Importantly, the mean sweat chloride in patients treated with triple therapy decreased from 102 to 58 mmol/L, i.e. below diagnostic threshold of 60 mmol/L. BMI also improved sig- nificantly at week 24, with a mean treatment difference of 1.04 relative to placebo. Serious adverse events occurred in 28 patients (13.9%) in the triple therapy arm and 42 patients (20.9%) in the placebo arm. There were no deaths in either trial group. Two patients (1.0%) in the triple therapy group discontinued the trial regimen because of adverse events, while no patients in the placebo group discontinued the trial regimen because of an adverse event. Overall, the triple therapy was generally safe with an acceptable side-effect profile, findings that are consistent with those of the phase 2 trial of elexacaftor–tezacaftor–ivacaftor [69].These data need to be confirmed in the long term. A 96-week open-label study of the triple combination elexacaftor–tezacaf- tor–ivacaftor regimen in people with CF who are homozygous or heterozygous for F508del is ongoing (NCT03525574).

4.4. Investigational end points and novel biomarkers

Given the complexity of pathophysiology, other endpoints more sensitive than lung function may accelerate drug development and testing. In this context, biomarkers, considered as ‘a char- acteristic that is objectively measured and evaluated as an indi- cator of normal biological processes, pathogenic processes, or biological responses to a therapeutic intervention’ [129], are becoming recognized as a critical tool for CF drug development [130]. In particular, young CF patients who typically have both mild disease and poorly standardized outcome measure may benefit from the development of more sensitive disease biomar- kers. As previously mentioned, LCI is emerging as a useful tool for detecting early lung disease in young people with CF [131]. Other studies have focused on the evaluation of CFTR as a chloride channel and ion transport in vivo.

Recently, to test the efficacy of ivacaftor–lumacaftor not only clinical outcomes were considered but also CFTR biomarkers including sweat-chloride concentration, nasal potential difference (NPD) measurement, and intestinal current mea- surement (ICM) in a short-term period (8–16 weeks) in patients homozygous for F508del aged 12 years and older [132]. This study was not placebo-controlled and did not assess any safety issues. A significant improvement of ppFEV1 and BMI similar to that obtained in the previous phase 3 clinical trials [71] was documented, and was paral- leled by partial rescue of F508del function: sweat chloride was reduced by 17.8 mmol/L, total chloride conductance in NPD showed levels corresponding to 10.2% of normal CFTR activ- ity, and ICM showed functional rescue of CFTR to a level of 17.7% of normal. Although these investigational endpoints did not correlate with changes in ppFEV1 or BMI, they indicate that upon lumacaftor–ivacaftor treatment they were changed to levels comparable to the lower range of CFTR activity in patients harboring CFTR residual function mutation. Longitudinal studies are needed to evaluate the impact of rescue to 10–20% of normal CFTR activity on long-term clin- ical outcomes and survival in CF people homozygous for F508del.

Blood-borne markers of inflammation may be extremely useful, since blood draws are repeatable, and can be obtained from subjects of any age and disease severity. Systemic inflammation may also link pulmonary and non- pulmonary CF comorbidities. In CF, C-reactive protein, serum amyloid A, calprotectin, neutrophil elastase- antiprotease complexes, various cytokines, and circulating mononuclear RNA transcripts have been evaluated as promis- ing candidates [130]. Although their usefulness has been established for gauging the treatment of pulmonary exacer- bations [133], few studies on CFTR modulator therapies have been conducted on neutrophils and mononuclear cells. Pohl and colleagues demonstrated that ivacaftor treatment resulted in normalization of defective neutrophil degranulation both in in vitro studies and in neutrophils obtained from clinically stable CF patients with the F508del/G551D genotype receiving 150 mg ivacaftor twice daily [134]. Others have found that ivacaftor treatment for 6 months of patients bear- ing one G551D mutation determined a decrease, toward normalization, of the activation status of blood leukocytes in vivo, as judged by the expression levels of CD63 (a marker of degranulation), CD11b, and intracellular activity of the inflammasome-associated enzyme caspase-1, which leads to the production of the potent pro-inflammatory cytokine interleukin-1β [135]. A more recent study observed that plasma donated by F508del/G551D heterozygote patients on ivacaftor therapy had reduced levels of the chemokines CXCL7, CXCL8, as well as of the cytokine TNF-α, similar to non-CF plasma levels [136]. These results were paralleled by those obtained in circulating neutrophils, where it was noted an increased membrane cholesterol content and a reduced cell adhesion, indicating the anti-inflammatory effects of iva- caftor treatment.

In our own experience, we have investigated the CFTR activity ex vivo in mononuclear cells (MNC) drawn from patients with at least one non-G551D gating mutation, finding that the CFTR-dependent chloride efflux (as studied by spec- trophotometry) was significantly increased after 24 weeks of ivacaftor treatment [137]. The correlation between MNC chlor- ide efflux with ppFEV1, indicates that this test could be a surrogate marker similar to the respiratory function para- meters. Recently, we monitored MNC CFTR-dependent chlor- ide efflux in patients homozygous for F508del before and after treatment with lumacaftor–ivacaftor. CFTR-dependent chloride efflux was not detectable in MNCs from CF patients prior to lumacaftor–ivacaftor treatment. Cumulative data obtained from nine patients showed that the CFTR-dependent chloride efflux in MNCs was significantly rescued only after 24 weeks post-treatment [138]. Since we have already shown that this assay is also sensitive to antibiotic treatment [139], it could become a promising surrogate noninvasive endpoint for the evaluation of CFTR activity in vivo. Blood mononuclear cells are also being evaluated for the impact of CFTR modulators at the level of gene expression. Sun and colleagues intriguingly found profound transcriptomic impacts of ivacaftor in MNC from patients with CF with at least one G551D allele, and that a panel of signature genes was useful for predicting clinical responsiveness to ivacaftor before treatment [140].Overall, these studies highlight the potential of leukocyte phenotypic and activity outcomes as useful parameters to be examined in future clinical trials utilizing CFTR modulators.

5. Marketing and post-marketing

The treatments based on CFTR modulators are extremely expensive and affect an already high health expenditure for CF patients, as the annual patient cost of ivacaftor, lumacaf- tor–ivacaftor, or tezacaftor–ivacaftor is approximately 258 USD,000–300,000 for each single drug [141]. While the scien- tific community is trying to expand the use of CFTR modula- tors to other class mutations and age groups, these costs are clearly not sustainable by the health-care systems of many countries, especially those with universal care coverage [142]. Recently, a pharmacoeconomic evaluation of lumacaftor-iva- caftor conducted in the US warned that the incremental cost-effectiveness ratio (ICER), defined as the difference in the ratio of cost per FEV1% predicted of lumacaftor–ivacaftor and pla- cebo, is of 95,016 USD [143]. The limit of this study is that these results may not be generalizable beyond a 1-year time horizon, especially if serious adverse events associated with lumacaftor-ivacaftor play a role in influencing the overall cost. In this context, the post-marketing analysis of tezacaftor, iva- caftor, and elexacaftor is urgently awaited. Like Orkambi®, rapid sales growth is predicted for Symdeko and triplet VX- 445–tezacaftor–ivacaftor, with consensus forecast sales of 2.167 USD billion and 2.226 USD billion, respectively, antici- pated for 2023 [144]. Up to now, Vertex holds a monopoly within the CFTR modulator market; however, other companies are conducting early phase studies of CFTR modulators, including Galapagos-AbbVie, Novartis, Proteostasis, and Flately [144,145]. The discovery of novel CF disease- modifying therapies, including co-potentiators and other ther- apeutic strategies based on gene editing, will likely lead to a reduction in costs for these etiologic therapies.

Recent studies are considering the clinical efficacy of the combination therapies in a real-world clinical practice and by investigational biomarkers. Popowicz and colleagues have reported that CF patients with deteriorated lung function (ppFEV1 < 40) upon lumacaftor–ivacaftor treatment experienced a decline in ppFEV1 from baseline at 2-h post initiation that persisted at 24 h but recovered in most patients at 1-month [146]. Another observational study in CF patients homozygous for F508del and severe lung disease reported that respiratory adverse events (AEs) occurred in 27 of 53 subjects (51%) and 16 (30%) discontinued treatment [116]. However, ppFEV1 improve- ments over 3 months of treatment were comparable to those observed in phase 3 studies. As to extrapulmonary effects, no significant change in BMI was observed within 3 months of treatment with lumacaftor–ivacaftor. In another study, the com- bination of lumacaftor–ivacaftor were measured at baseline and 8–16 weeks after initiation of therapy with the approved dose of lumacaftor 400 mg in combination with ivacaftor 250 mg every 12 h [132]. This window for follow-up measurements was selected to facilitate scheduling of study visits in a real-life setting based on the results of the pivotal phase 3 study showing sustained effects of lumacaftor–ivacaftor on clinical outcomes at Week 8 and Week 16, and phase 2 and recent pediatric phase 3 studies demonstrating sustained effects on sweat chlor- ide from Week 2 through Week 24 [71,110,111,147]. They found functional correction by the three CFTR biomarkers sweat chlor- ide, NPD, and ICM, although no correlation was found between improvement of F508del-CFTR function and FEV1% predicted or BMI. Others have reported the results of a retrospective cohort study of CF individuals who initiated treatment with lumacaftor/ ivacaftor outside a clinical trial. Participants (adults and children; mean age: 24.7 years; range: 12–59 years) were homozygous for F508del and were followed from 1 year before drug initiation to up to 11 months post initiation [148]. Forty-six subjects (39.7%) reported adverse effects related to lumacaftor–ivacaftor, with the vast majority (82.2%) being pulmonary adverse effects, and 20 subjects (17.2%) discontinued lumacaftor–ivacaftor because of adverse effects. Finally, a nationwide study of efficacy and safety in a cohort of adolescents and adults homozygous for F508del revealed that ivacaftor–lumacaftor was discontinued in 18.2% of patients, mostly due to respiratory AEs and, to a lesser extent, to nonrespiratory AEs. Patients who received the combination ther- apy for up 1 year showed improvements in lung function and nutritional status [149]. Overall, these results anticipate what would be the evaluation of dual therapy ivacaftor–tezacaftor in post-marketing studies, i.e. with patients who often show reduced lung function and less stable disease characterized by higher rates of exacerbations than those included in clinical trials. In general, many studies have reported beneficial extrapulmon- ary effects related to the use of ivacaftor in patients with CF with at least one gating mutation, including weight gain and growth, pancreatic function, endocrine function, intestinal and hepatobili- ary function, bone disease and physical activity, and fertility, most of the evidence is low or very low quality, given the limited number of patients evaluated and the lack of control groups [150]. On the other hand, ivacaftor–lumacaftor therapy has, overall, failed to demonstrate major extrapulmonary benefits, due to the limited studies performed, not to speak for ivacaftor–tezacaftor which will be evaluated for extrapulmonary effects as soon as placebo-controlled clinical trials will be executed. In summary, longer real-life evaluation of combination therapies based on ivacaftor and tezacaftor has to be war- ranted to examine their safety profile as well as their effec- tiveness in preventing lung function decline and pulmonary exacerbations. More precise and affordable direct and surro- gate biomarkers are also needed to better gauge the effec- tiveness of tezacaftor–ivacaftor in triple therapy. 6. Conclusions The clinical benefit of ivacaftor in CF patients with G551D mutation is considered to be the benchmark for treatment with highly effective CFTR modulators. Given the complexity of multiple defects associated with the F508del mutation, the achievement of a clinically relevant outcome in patients homozygous for F508del has been more difficult to achieve and needed the association of drugs with different mechan- isms of action. Although the combination therapy of the potentiator ivacaftor and a corrector molecule has advanced the treatment of CF patients homozygous for the F508del mutation and also for those heterozygous for F508del and a MF mutation, the combination tezacaftor–ivacaftor shows some advantages over lumacaftor–ivacaftor. No discontinua- tion of tezacaftor–ivacaftor was observed due to severe respiratory adverse effects. Moreover, this dual therapy has a more favorable medications interaction profile. A prospective observational single-center trial is recruiting F508del homozygous patients to observe the effect of chan- ging from lumacaftor–ivacaftor to tezacaftor–ivacaftor in order to ameliorate the safety profile (TRANSITION study; ClinicalTrials.gov identifier: NCT03445793). Finally, the intro- duction of triple therapy with elexacaftor–tezacaftor–ivacaftor is expected to lead to meaningful improvements in the lives of individuals with CF homozygous or heterozygous for the F508del mutation, encompassing almost the totality of this population. It is anticipated that in the future CF will no longer be the most common lethal autosomal recessive disorder in Caucasian individuals, but a chronic disease with a normal life expectancy. 7. Expert opinion The lack of a reliable animal models of CF has pushed the scientific community to search for alternative models in order to judge the effectiveness of a new lead compound. However, the transposition from in vitro data to the in vivo clinical setting as to the application of CFTR modulators in CF is not without shadows. Classical cellular models cannot recapitulate the complexity of physiological activity and regulation of CFTR, a regulator of other channels and of other tissue-based processes, such as innate immunity, at the mucosal level. Novel and sustainable CF patient-specific tissue models have to be better pursued in order to gain real-life assumption on CFTR-modulator therapies and observe a smaller gap when these molecules are applied to CF individuals. Recently, it has been found that CFTR residual function and responses to drug therapy in rectal organoids depended on both the CFTR muta- tion and the genetic background of the subjects. On the other hand, it is also true that the native airway epithelium may represent a challenge for these studies: it is damaged in vivo and epigenetic changes could alter the drug response. An effort in developing patient-specific lung-derived organoids should be pursued. Induced pluripotent stem cells may help in raising lung organoids specific for a single CF individual. Some encouraging results on the correlation between assay- based rectal organoids or HNE cells and clinical end points, such as ppFEV1 and sweat chloride concentration, have been provided for ivacaftor or the association lumacaftor–ivacaftor, paving the way in the direction of testing tezacaftor and triple associations. These findings support the potential use of rectal organoids and HNE to study novel compounds targeting the primary defects caused by CF-causing mutations. Moreover, they could be used to determine the basic for the variable drug responses observed amongst individuals treated with CFTR modulators. Rectal or lung biopsies are invasive, making possible that repeated measures during a drug treatment would be less feasible. Noninvasive secretion-derived (e.g. from sputum) or blood-borne markers may address this limitation. Established biomarkers, such as elastase or antiprotease-elastase com- plexes, should be further prospectically assessed in their valid- ity in patients which will be treated with different combinations of tezacaftor–ivacaftor and other compounds, not only in CF people with one allele bearing the F508del mutation, but also in those individuals with 1-single patient mutations. Later in this year, FDA has approved Trikafta™ (elexacaftor/ tezacaftor/ivacaftor) for the treatment of CF in people ages 12 years and older who have at least one F508del mutation (~6,000 individuals). This triple therapy may thus be extended to off-label indications, e.g. in the case of a rare residual function mutation or a class I stop mutation in combination with inhibitors of nonsense-mediated mRNA decay or read- through drugs. In clinical trials, Trikafta™ was shown to be safe and effective with potentially fewer negative side effects than previous modulators. However, it has to be mentioned that this triple therapy comes with warnings related to ele- vated liver function tests, drug–drug interactions with pro- ducts that are inducers or inhibitors of a certain liver enzyme (CYP 3A), and the risk of cataracts in younger age groups. Moreover, certain drugs may interact with Trikafta™, includ- ing some antifungal medicines and some antibiotics. Triple therapy is not recommended if the patients is on certain antibiotics (rifampin or rifabutin), specific seizure medica- tions, or St. John’s Wort. Under these considerations, an advancement in other CFTR modulators without these inter- actions might guarantee the application of triple therapy in those requiring antifungal or antituberculosis mycobacterial therapy. In the next 5 years, it is likely that CF practitioners will experience that initiation of treatment for CF patients will occur simply by use of biomarkers of CFTR expression (eg. sweat chloride, nasal potential difference, rectal organoids) rather than testing for specific mutations. Overall, tezacaftor– ivacaftor as double or triple therapy may be successfully applied also in those patients harboring rare uncharacterized CFTR mutations. Finally, CFTR modulators will be tailored to individuals based on their cell culture response using a process called ‘theratyping’, meaning that CFTR variants can be classified according to their effect on the CFTR protein and their responsiveness to CFTR modulators.